One of the most widely studied and accepted procedures in clinical chemistry is the determination of serum and urine .alpha.-amylase which is used for the diagnosis of pancreatic disease.
During the past twenty-five years various amylase methods have been developed for use in the clinical laboratory. Some of the methods, i.e. saccharogenic method, involves complicated methodology which makes its routine use prohibitive. Other methods, i.e., turbidometric and viscosimetric methods for the determination of .alpha.-amylase activity are dependent on changes in the physical properties of the substrate, which may be influenced to a considerable degree by other factors present in the serum. Today, one of the most widely used methods for .alpha.-amylase determination is the starch-iodine method. With this method only a specific portion of the substrate is measured and the enzyme does not work under substrate saturation conditions. Further, the presence of serum proteins could interfere with the starch-iodine reaction.
In addition to the above difficulties associated with the mentioned methods, a further difficulty is encountered because the aforementioned methods can be used to determine a rather limited range of .alpha.-amylase activity. Also, some of the methods cannot be used for accurate determination of either sub-normal or highly elevated .alpha.-amylase levels.
It has been shown that .alpha.-amylase activity can be determined based on the solubilization of colored starch grains by .alpha.-amylase. It has also been shown that by using cross-linked soluble starch marked with a dye marker under alkaline conditions, .alpha.-amylase activity could be determined. However, these reported procedures require elevated incubation temperature, prolonged incubation time and oftentimes double dialysis to achieve adequate sensitivity.
Synthetic fluorogenic and chromogenic starch substrates have also been used to determine amylase activity in biological fluids. In these methods a chromophore is covalently bound to a water insoluble, cross-linked starch. Amylase acts upon the substrate to release water-soluble fragments which are measured spectroscopically after first separating the water-insoluble residue.
In another assay involving a synthetic substrate (Nature, 182 (1958) 525-526) a p-nitrophenol derivative of maltose is used. The p-nitrophenol replaces the anomeric hydroxyl group of maltose. Amylase causes cleavage of the substrate to produce p-nitrophenol which can be monitored at 410 mm. However, the assay is 16 hours long and maltase also cleaves the substrate. In addition, since this derivative has no endo-.alpha.-1,4 bond its specificity as an amylase substrate is subject to considerable questions.
The method according to the invention is distinguished from the known prior methods by either greater simplicity, greatly decreased incubation time of greater sensitivity. Further, the method of the present invention is distinguished by using a controlled molecular weight polysaccharide in place of maltose. These controlled molecular weight polysaccharides tagged with p-nitrophenol, while having sufficient chain length to resist cleavage by maltase, and possessing an endo 1,4 bond can still react rapidly with amylase.